The present invention relates generally to atomic force microscopy and more particularly relates to an atomic force microscope and controller which minimize contact forces between a probe tip and a specimen and is well suited for the study of biological specimens.
In the study of biology, it is desirable to observe biological specimens under very high magnification in a native environment. Such observations allow scientists to monitor, in real time, biological processes at the molecular and sub-molecular level. Such processes include the interaction of proteins with DNA and with each other. Currently, these processes cannot be observed in real time with electron microscopes or x-ray crystallography techniques which are known in the art, as the specimens are not in their native environment when using these apparatuses. Accordingly, scientists have sought alternate methods to observe biological specimens. One such alternative is known as the atomic force microscope.
Atomic force microscopes (AFM), which are generally known in the art, physically probe a specimen to create an image of the specimen's surface. FIG. 1 illustrates a typical embodiment of an AFM known in the art. The AFM has two primary components, a scanner 10 and a flexible cantilever 12 having a probe tip 14 on a free end. The scanner 10 has a top surface 16 on which a specimen 18 to be imaged is placed. The scanner 10 typically employs three piezoelectric elements 20, 22, 24 to move the specimen 18 in three dimensions, X Y and Z, relative to the position of the probe tip 14. The probe tip 14 is affixed to the free end of the flexible cantilever 12 and contacts the specimen 18. The AFM includes a laser 26 directed onto the cantilever 12 and a photo detector 28 which is responsive to laser light to measure the deflection of the cantilever 12. As the degree of cantilever deflection is proportional to the contacting force between the probe tip 14 and the specimen 18, such force can accurately be calculated based on the angle of cantilever deflection.
To create an image of a specimen, the scanner 10 directs the specimen 18 in a raster-scan fashion in the X-Y direction while continuously sampling the contour of the specimen 18 in the Z direction. The sampling is generally performed using one of two techniques known in the art, namely contact mode or tapping mode. In contact mode, the scanner 10 is controlled in the Z direction such that the contacting force between the probe tip 14 and the specimen 18 is substantially constant. As the contour of the specimen changes, the deflection of the cantilever 12 also changes and a servo system driving the scanner 10 adjusts the Z coordinate of the scanner 10 to restore the desired constant force. At each specimen point, the coordinate of the Z axis is indicative of the specimen contour. Because the probe is constantly contacting (i.e. it drags along) the surface of the specimen during the X-Y raster scan, significant lateral forces are applied to both the specimen 18 and the probe tip 14. The probe tip 14, which is typically 200–300 Angstroms in diameter is subject to rapid wear and breakage under these forces. Also, when used on soft specimens, such as biological specimens, the probe tip is likely to destroy the surface of the specimen, making accurate and repeatable measurements impossible.
In tapping mode, the cantilever 12 is driven in an oscillatory fashion at the resonant frequency of the cantilever. This may be achieved by affixing the cantilever to a piezoelectric element 30 and driving the piezoelectric element 30 with a voltage signal at the resonant frequency of the cantilever. To determine the contour of the specimen in tapping mode, the scanner 10 moves the specimen in the Z direction until a predetermined reduction in oscillation amplitude is detected. The reduction in oscillation amplitude is the result of the probe tip 14 contacting the surface of the specimen 18 during each cycle of oscillation. Because the probe tip 14 repeatedly, but only momentarily contacts the specimen 18 at each point during the X-Y raster scan, the lateral force present during contact mode is substantially reduced. However, because the probe tip 14 is moving rapidly on arrival at the specimen surface, the contacting force, while short in duration, is large in magnitude. The force that results from tapping mode tends to be destructive to biological specimens. Thus, tapping mode is most useful in sampling hard surfaces, such as those found in integrated circuit manufacturing processes and the like. Also, tapping mode is difficult to use when measuring a fluid-based specimen. When the cantilever assembly is submerged into a fluid environment, the desired oscillation of the cantilever can be dampened and additional resonances are developed which can adversely affect operation and accuracy. Also, fluid flow induced by the tapping oscillation tends to erode the specimen. Because biological specimens tend to reside in a fluid environment, tapping mode is not well suited for measuring these specimens. Tapping mode is also incapable of separately measuring the contacting force and adhesion force at each pixel. Because tapping mode cannot separate the two forces, concurrent mapping of each force during scanning is presently not possible with AFM's employing tapping mode.
An alternative operating mode to both contact mode and tapping mode is described in U.S. Pat. No. 5,229,606 to Elings et al. Elings et al. refers to “jump scanning” where the probe is momentarily brought into contact with the surface to be measured. The probe is then lifted away from the surface as the specimen is moved in the X direction and the probe tip is then brought back down into contact to take the next specimen. By jumping over the surface of the specimen, Elings et al. teach a method of increasing scanning speed with reduced risk of probe damage. However, when the probe tip and specimen contact one another, an attractive force tends to hold the probe tip in contact with the specimen. To ensure that the probe tip is able to release, the cantilever 12 must be formed with a sufficient spring constant to overcome this attractive force. Unfortunately, increasing the spring constant of the cantilever 12 increases the magnitude of the contact force between the probe tip 14 and specimen 18 which is required to achieve a measurable cantilever 12 deflection. Such stiff cantilevers, i.e., in the range greater than 0.1 Newtons per meter (N/m), which are required for jump mode, are incompatible with the more sensitive biological specimens which are easily damaged under the application of such forces.
The problem of overcoming the attractive forces between an AFM probe tip 14 and specimen surface was addressed in U.S. Pat. No. 5,515,719 to Lindsay. Lindsay operates in a mode where contact between the probe tip 14 and specimen surface is considered to be undesirable and recognized that when soft (low spring constant) cantilevers are used, the attractive interaction between the specimen 18 and probe tip 14 tends to draw the probe tip in and the probe tip 14 will contact and stick to the surface until enough force is applied to the cantilever base to release the probe tip 14. To address this problem, Lindsay teaches the addition of a magnetic particle attached to the cantilever in combination with a magnetic solenoid located proximate to the cantilever. The solenoid generates a magnetic field which is variable and precisely regulated by a servo circuit. The servo circuit monitors the deflection of the cantilever and continuously adjusts the magnetic field such that the attractive force between the probe tip 14 and specimen 18 is substantially neutralized. In this way, the probe tip 14, as taught by Lindsay, is kept at a distance from the specimen and never makes contact with the specimen. Therefore, it is impossible to measure an adhesion force using this method and difficult to measure the specimen profile at high lateral resolution because of the tip-specimen separation.
The use of a magnetic particle affixed to a flexible cantilever and controlled by a magnetic coil as used by Lindsay was first disclosed in an article by Florin et al., entitled “Atomic Force Microscope with Magnetic Force Modulation”, published in the Review of Scientific Instrument, 65(3), March 1994. Florin et al. teach the use of a magnetic control system to drive the cantilever in an oscillating fashion such that the probe tip momentarily and adversely contacts a specimen, in a manner similar to tapping mode.
Current AFM techniques tend to be destructive to biological specimens. Therefore, there remains a need for an improved atomic force microscope adapted for use in a fluid medium: for the observation of biological specimens in their native environment under conditions completely controlling and quantitatively measuring the interaction forces between tip and specimen.